Herpes Simplex Virus Type 1 Thymidine Kinase Expression Research Articles

Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population.

We have stably expressed in CD4+ lymphoid cells the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene under the control of the human immunodeficiency virus (HIV) promoter and transactivation response element sequences. Upon HIV infection these regulatory sequences were transactivated, switching on high-level expression of HSV1-TK. This in turn caused the death of HIV-infected cells when they were cultured in the presence of acyclovir, a nucleoside analog that becomes toxic after phosphorylation by HSV1-TK. The elimination of HIV-infected cells resulted in the arrest of HIV spreading in the culture. Complete protection of HSV1-TK-expressing cells was obtained using acyclovir concentrations that are commonly detected in the plasma of patients treated for HSV1 infection. Thus, expression of this DNA construct generates a pool of CD4+ booby-trapped cells that, as a population, are resistant to HIV infection. Our data provide a rationale for the use of suicide genes in the design of gene therapy of HIV infection.

The Herpes Simplex Virus Type 1 Thymidine Kinase Is Expressed in the Testes of Transgenic Mice under the Control of a Cryptic Promoter

We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box-independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia.

The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter.

We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box-independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia.

Presence of DNA synthesis and mitosis in the brain of young adult mice

Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.